Vector-mediated transgene delivery finds utility in the treatment of genetic disorders by supplementing a protein or other substance which, is either absent, or present in insufficient amounts in the host. Adenoviral (Ad) vectors are highly efficient vehicles for transgene delivery. Adenoviral-based gene-transfer vectors have a number of features that make them particularly useful for gene transfer into cells including the fact that the biology of adenovirus is well characterized, adenovirus is not associated with any known human disease, adenovirus is efficient in introducing its DNA into host cells, the virus has a broad host cell range and large scale production has been accomplished. Human adenoviral-based vectors, in which at least the E1 region has been deleted and replaced by a gene of interest have been used extensively for gene therapy. Adenovirus vectors currently used in gene therapy are typically replication incompetent and have a deletion in the E1 region.
The features which make recombinant adenoviruses potentially powerful gene delivery vectors have been extensively reviewed (Berkner, Biotechniques 6: 616–629, (1988) and Kozarsky & Wilson, Curr. Opin. Genet Dev. 3: 499–503, (1993)). Controlled replication of adenoviral vectors, Whether through gene deletion or replication restricted to particular tissues, is of particular importance for in vivo applications involving adenovirus.
Replicative adenoviruses have been engineered to achieve selective targeting and amplification in vivo. Conditionally replicative and oncolytic adenoviruses have shown great promise in the treatment of cancer (Yu et al., Curr. Opin. Mol. Ther. 2002, Oct; 4(5):435–43, Bell et al., Curr. Gene Ther. 2002 May 2(2):243–54; Yoon et al. Curr. Cancer Drug Targets 2002 August; 1(2):85–107). Replicative adenoviruses can be delivered systemically, can be targeted to tumor cells, and can amplify their cytolytic effect in a tumor-specific manner, thereby providing substantial clinical benefit. See Henderson et al., U.S. Pat. No. 5,698,443; Hallenbeck et al., WO 96/17053. In such systems, a cell-specific transcriptional regulatory element controls the expression of a gene essential for viral replication, and thus, viral replication is limited to a cell population in which the element is functional. For example, an attenuated, replication-competent adenovirus has been generated by inserting the prostate-specific antigen (PSA) promoter and enhancer (PSE-TRE) upstream of the E1A transcription unit in adenovirus serotype 5 (Ad5), which virus demonstrates selective cytotoxicity toward PSA expressing cells in vitro and in vivo (Rodriguez et al. (1997) Cancer Res. 57:2559–2563).
Adenovirus of interest, including oncolytic adenovirus, conditionally replicative adenovirus, and replication defective adenovirus are frequently engineered to have genetic modifications in the E1 early gene region (genetic map units 1.30 to 9.24) of the virus genome. Typical modifications include deletions within the E1 gene region and/or replacement of the E1A promoter, introduction of a transgene, etc. Helper virus-independent production of adenovirus can require a packaging cell line that complements for viral gene products.
In order to produce recombinant adenoviral vectors for research and clinical trials, a packaging cell line is transfected with adenoviral E1 coding sequences. The cell line must express sufficient E1 gene products to supply in trans the E1A and E1B gene products that are required directly and indirectly for adenoviral DNA replication and virion production.
Although E1 complementation permits the production of recombinant adenoviral vectors, recombination events between the transfected E1 sequences in the host cell and the adenoviral vector can occur, resulting in the generation of replication competent adenovirus (RCA). This is especially problematic with large-scale production and successive propagation, and hence is problematic in the preparation of adenoviral particle stocks for therapeutic uses. Recombination and the development of RCA during recombinant adenoviral vector production not only contaminates viral stocks, but also is problematic relative to use of adenoviral vectors for in vivo applications. The problem of RCA generation has been known for some time, as described for example in Shenk et al., 1979, Cold Springs Harb. Symp. Quant. Biol. 44 (1979) 367–375 and Lochmuller, Human Gene Therapy, 1994, 1485–1491.
Available packaging cell lines typically contain adenoviral genes that have been deleted from the vector but are required for viral replication. In some cases overlapping sequences between the host cell and adenoviral vector are not completely eliminated. For example, the human embryonic kidney derived 293 cells (Graham et al. (1977) J. General Virology 36:59–74) have been widely used for propagating adenoviral vectors. However, due to substantial overlapping sequences between the adenoviral vector genome and the 293 cell line, recombination events occur that result in the generation of a replication competent adenoviral particles.
Improvements have been made to reduce the possibility of generating replication competent vectors due to recombination events between the packaging cell line and the vector via reduction in the sequences common to the vector and cell line (Fallaux et al. (1998) Human Gene Therapy 9:1909–1917). For example, U.S. Pat. No. 5,994,128 describes cell lines that complement for both E1A and/or E1B, while retaining the natural E1B promoter sequences. Studies performed using the PER.C6 cell line demonstrated that, despite a single region of homology between this cell line and the adenoviral vector, RCA were generated and cytopathic effects were observed in a cell based assay (Kim et al. (2001) Exp. Mol. Med. 33(3)145–9). When analyzed, the RCA were shown to contain the PGK promoter-E1 gene, derived from the plasmid that was employed to construct the PER.C6 cell line. The same problem of residual sequence overlap is true of other cell lines developed as alternatives to 293 cells. (See, for example, Massie et al., U.S. Pat. No. 5,891,690; Kovesdi et al., WO 95/34671, Kedan et al., PCT/US95/15947, Schiedner et al. (2002) Human Gene Therapy, 11:2105–2116). Consequently, there remains the potential for unwanted recombination events between the cell line and the adenoviral vector.